The taxane family of terpenes is considered to be an exceptionally promising group of cancer chemotherapeutic agents. Many taxane derivatives, including paclitaxel, docetaxel, taxcultine canadensol are highly cytotoxic and possess strong in vivo activities in a number of leukemic and other tumor systems. Paclitaxel, and a number of its derivatives, have been shown to be effective against advanced breast and ovarian cancers in clinical trials (W. P. MacGuire et al., Annals of Internal Medicine, vol 111, pg. 273, 1989). They have also exhibited promising activity against a number of other tumor types in preliminary investigations. Paclitaxel has recently been approved in the U.S. and Canada for the treatment of ovarian cancers (Rose et al., in "The Alkaloids", A Brossi, Ed., Academic Press, New York, Paclitaxel: A Review of its preclinical in vivo Antitumor Activity. Anti-Cancer Drugs 3, 311-321 1992; and Suffness, M., Paclitaxel: from discovery to therapeutic use. Ann. Rep. In Med. Chem, 28, 305-314, 1993). Taxanes are believed to exert their antiproliferative effect by inducing tubulin polymerization, which forms extremely stable and nonfunctional microtubules (Schiff, et al., Promotion of Microtubule Assembly in vitro by Paclitaxel. Nature, 277, 665-667, 1979). However, a major problem with the clinical studies is the limited availability of paclitaxel and its derivatives.
Taxanes are natural products which can be isolated from yew trees. The first taxane to be characterized was paclitaxel (also known as taxol.TM.) which was isolated and purified from the bark of the Pacific yew in 1971. The only available natural source of paclitaxel to date are several species of a slow growing yew (genus Taxus), wherein paclitaxel is found in very low concentrations (less than 400 parts per million) in these trees. Furthermore the extraction is difficult, the process is expensive and the yield of paclitaxel is low (Huang el al, J. Nat. Prod. 49 665, 1986, reported a yield of 0.00025% of a crude paclitaxel fraction from Taxus brevifolia bark). ##STR1##
Paclitaxel can be isolated from the bark of Taxus brevifolia, the pacific yew tree, or from Taxus baccata, its European relative. Since removal of the bark destroys the trees and endangers the species, isolation of taxanes from the stems and needles of various Taxus species offers hope that the supply of taxanes, in particular paclitaxel, would become more abundant.
The preparation of paclitaxel derivatives, some of which have been reported to demonstrate enhanced chemotherapeutic activity, ultimately depends upon the supply of the parent compound--baccatin III. The structure of baccatin III has the basic diterpenoid structure of paclitaxel without the side chain at the C-13 position. ##STR2##
Baccatin III is an important starting material in paclitaxel semi-synthesis. Therefore the significance of baccatin III will likely increase as more clinical studies are performed using paclitaxel. One such reason is that it appears that water soluble paclitaxel-like compounds with slightly modified C-13 side chains may be more desirable as cancer chemotherapeutic agents than the naturally occurring less water soluble paclitaxel. This increases the urgent need for baccatin III as a starting material to synthesize both paclitaxel and second or third generation paclitaxel-like compounds. There is, therefore, a need for an improved method of isolating and/or synthesizing Baccatin III.
The majority of research to date has been concerned with the development of techniques to increase the availability of either paclitaxel or baccatin III. These techniques have included improvements to the isolation and purification processes (U.S. Pat. No. 5,407,674 and U.S. Pat. No. 5,380,916), to the total synthesis (U.S. Pat. No. 5,405,972) and partial synthesis (from more abundant paclitaxel precursors) and also isolation from a variety of cell culture systems U.S. Pat. No. 5,019,504). In Addition, an endophytic fungi isolated form bald cypress (Taxodium distichum) was reported to produce very small amounts of paclitaxel (Strobel, R. et al., Microbiology, 142, 2223-2226, 1996)
Because of the structural complexity of paclitaxel, partial synthesis is a far more viable approach to providing adequate supplies of paclitaxel and paclitaxel precursors than total synthesis. The first successful semi-synthesis of paclitaxel was developed by Denis et al, (U.S. Pat. No. 4,924,011 re-issued as U.S. Pat. No. 34,277), using the starting material 10-deacetylbaccatin III which can be extracted in relatively high yield from the needles of specific species. ##STR3##
In fact, most of the research to date regarding the semi-synthesis of paclitaxel has involved 10-deacetylbaccatin III. The conversion of 10-deacetylbaccatin III into paclitaxel is typically achieved by protecting the hydroxy at C-7, attachment of an acetyl group at the C-10 position, attachment of a C-13 .beta.-amido ester side chain at the C-13 position through esterification of the C-13 alcohol with the .beta.-lactam moiety, and deprotecting C-7. Since the supply of 10-deacetylbaccatin III is limited, other sources should be pursued.
Research has recently centred on semi-synthesis of paclitaxel from 10-deacetylbaccatin III because it is the major metabolite obtained from specific species of the European Yew (Taxes baccata). However to date, the yields of 10-deacetylbaccatin III have been unsatisfactory, ranging from 50-165 mg taxane per kilogram of starting material (i.e. providing yields of between 0.005 to 0.017%). Hence there is an urgent need for novel semi-synthetic techniques to produce higher yields of paclitaxel precursors, such as baccatin III, for subsequent use in the production of paclitaxel derivatives. The present invention provides such a method, describing the conversion of a known taxane (9-dihydro-13-acetylbaccatin III), which is produced as a major metabolite in a certain species of taxus, into a paclitaxel precursor which produces relatively large amounts of a 7-protected baccatin III. Depending on the collection sites, the yield of 9-dihydro-13-acetylbaccatin III can vary from 2.0 to 2.5 g per kilogram of dry plant and this taxane can be chemically transformed, by the present invention, into 7-protected baccatin III in 20% yield.